PROJECT SUMMARY Nearly 600,000 patients are diagnosed each year with oral cancer worldwide, and a majority of these patients are treated initially with chemoradiation therapy because they present with locally advanced disease. Although radiation therapy typically lasts 6-7 weeks, tumor response is evaluated only 1-2 months after the end of therapy. An early determination of treatment resistance would greatly alleviate the pain and suffering for patients with treatment-resistant tumors who would otherwise undergo several weeks of ineffective therapy. In this proposal, we seek to leverage our lab's expertise and preliminary published and unpublished data demonstrating that tumor oxygenation and metabolism provide powerful biomarkers of treatment resistance. Our goal is to develop a non-invasive and quantitative tool that combines diffuse reflectance and Raman spectroscopy to reveal key metabolic, functional and molecular changes in response to radiation and chemotherapy in oral cancer. The integration of these two spectroscopic modalities is motivated by our preliminary data that shows significantly higher reoxygenation and elevated levels of key metabolites post-radiation in radiation-resistant tumors compared with sensitive tumors. The objective of this proposal is to develop, optimize, and `field-test' a multimodal spectroscopic probe for noninvasive sensing of treatment resistance in oral cancer. Because the majority of treatment-resistant oral cancers tend to be located in the larynx, the development and optimization of the probe will focus on enabling the probe's access through the working channel of a laryngoscope. This will require a small-diameter probe, which will in turn entail the use of short source-detector offsets. To enable spectral acquisition with high signal-to-noise and short acquisition times, we will develop an inverse spatially offset probe in Aim 1, and characterize spectral reproducibility and the effects of probe pressure. In Aim 2, we will investigate the dynamic changes in tumor oxygenation and metabolism after radiation and chemotherapy in vivo in known models of treatment resistance and sensitivity, and associate these optical markers with molecular biomarkers determined using immunohistochemistry. The multimodal probe will be tested in a pilot clinical study in Aim 3 to determine patient acceptance of daily measurements during chemoradiation therapy. In addition to establishing the ideal time points at which these measurements can be performed with minimal discomfort to patients, these studies will establish preliminary differences in optical biomarkers in treatment- resistant and sensitive patients that can be compared with our pre-clinical animal model results from Aim 2. Successful completion of these aims will delineate functional and molecular changes associated with radiation and chemoresistance at unprecedented time scales. This knowledge of radiobiological changes occurring immediately after therapy will not only aid in differentiating treatment responders and non-responders but also identify additional time points at which meaningful changes to therapy could improve treatment response rates.